The major hypothesis upon which this research is based is that non-collagenous components of mineralized connective tissues are intimately involved in the formation of the tissues. Furthermore, an understanding of the chemical composition, localization and regulatory mechanisms of biosynthesis are required before specific, functional roles can be elucidated. In this proposal we will address the aforementioned areas as they relate to proteoglycans and osteopontin, newly discovered bone-derived cell attachment protein. The approach taken is to isolate chemical amounts of these components, verify their purity and use them as antigens. The antibodies thus produced will be used in immunolocalization studies and as reagents in immunoassays. We will explore in detail the kinetics of proteoglycan biosynthesis in rat calvaria and long bone, in ROS 17/2.8 cells and in cultures of collagenase-derived rat calvarial osteoblasts. We will evaluate the role and mechanism that 1,25-dihydroxyvitamin D3 and parathyroid may have in the regulation of proteoglycan and osteopontin biosynthesis. We will also further explore the mechanism of action by which osteopontin stimulates cells to attach in vitro.